Risk of relapse of ANCA-associated vasculitis among patients homozygous for the proteinase 3 gene Val119Ile polymorphism.

BACKGROUND: The frequency of proteinase 3 gene (PRTN3) polymorphisms in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is not fully characterised. We hypothesise that the presence of a PRTN3 gene polymorphism (single nucleotide polymorphism (SNP) rs351111) is relevant for clinical outcomes. METHODS: DNA variant calling for SNP rs351111 (chr.19:844020, c.355G>A) in PRTN3 gene assessed the allelic frequency in patients with PR3-AAV included in the Rituximab in ANCA-Associated Vasculitis trial. This was followed by RNA-seq variant calling to characterise the mRNA expression. We compared clinical outcomes between patients homozygous for PRTN3-Ile119 or PRTN3-Val119. RESULTS: Whole blood samples for DNA calling were available in 188 patients. 75 patients with PR3-AAV had the allelic variant: 62 heterozygous PRTN3-Val119Ile and 13 homozygous for PRTN3-Ile119. RNA-seq was available for 89 patients and mRNA corresponding to the allelic variant was found in 32 patients with PR3-AAV: 25 heterozygous PRTN3-Val119Ile and 7 homozygous for PRTN3-Ile119. The agreement between the DNA calling results and mRNA expression of the 86 patients analysed by both methods was 100%. We compared the clinical outcomes of 64 patients with PR3-AAV: 51 homozygous for PRTN3-Val119 and 13 homozygous for PRTN3-Ile119. The frequency of severe flares at 18 months in homozygous PRTN3-Ile119 was significantly higher when compared with homozygous PRTN3-Val119 (46.2% vs 19.6%, p=0.048). Multivariate analysis identified homozygous PR3-Ile119 as main predictor of severe relapse (HR 4.67, 95% CI 1.16 to 18.86, p=0.030). CONCLUSION: In patients with PR3-AAV, homozygosity for PRTN3-Val119Ile polymorphism appears associated with higher frequency of severe relapse. Further studies are necessary to better understand the association of this observation with the risk of severe relapse.

Activation of a Latent Epitope Causing Differential Binding of Antineutrophil Cytoplasmic Antibodies to Proteinase 3.

OBJECTIVE: Proteinase 3 (PR3) is the major antigen for antineutrophil cytoplasmic antibodies (ANCAs) in the systemic autoimmune vasculitis, granulomatosis with polyangiitis (GPA). PR3-targeting ANCAs (PR3-ANCAs) recognize different epitopes on PR3. This study was undertaken to study the effect of mutations on PR3 antigenicity. METHODS: The recombinant PR3 variants, iPR3 (clinically used to detect PR3-ANCAs) and iHm5 (containing 3 point mutations in epitopes 1 and 5 generated for epitope mapping studies) immunoassays and serum samples from patients enrolled in ANCA-associated vasculitis (AAV) trials were used to screen for differential PR3-ANCA binding. A patient-derived monoclonal ANCA 518 (moANCA518) that selectively binds to iHm5 within the mutation-free epitope 3 and is distant from the point mutations of iHm5 was used as a gauge for remote epitope activation. Selective binding was determined using inhibition experiments. RESULTS: Rather than reduced binding of PR3-ANCAs to iHm5, we found substantially increased binding of the majority of PR3-ANCAs to iHm5 compared to iPR3. This differential binding of PR3-ANCA to iHm5 is similar to the selective moANCA518 binding to iHm5. Binding of iPR3 to monoclonal antibody MCPR3-2 also induced recognition by moANCA518. CONCLUSION: The preferential binding of PR3-ANCAs from patients, such as the selective binding of moANCA518 to iHm5, is conferred by increased antigenicity of epitope 3 on iHm5. This can also be induced on iPR3 when captured by monoclonal antibody MCPR2. This previously unrecognized characteristic of PR3-ANCA interactions with its target antigen has implications for studying antibody-mediated autoimmune diseases, understanding variable performance characteristics of immunoassays, and design of potential novel treatment approaches.

Autoreactive Plasmablasts After B Cell Depletion With Rituximab and Relapses in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: Autoreactive B cells are responsible for antineutrophil cytoplasmic antibody (ANCA) production in ANCA-associated vasculitis (AAV). Rituximab (RTX) depletes circulating B cells, including autoreactive B cells. We aimed to evaluate changes and associations with relapse of the circulating autoreactive B cell pool following therapeutic B cell depletion in AAV. METHODS: Sequential flow cytometry was performed on 148 samples of peripheral blood mononuclear cells from 23 patients with proteinase 3 (PR3)-ANCA-positive AAV who were treated with RTX for remission induction and monitored after stopping therapy during long-term follow-up in a prospective clinical trial. PR3 was used as a ligand to target autoreactive PR3-specific (PR3+) B cells. B cell recurrence was considered as the first blood sample with ≥10 B cells/µl after RTX treatment. RESULTS: At B cell recurrence, PR3+ B cell frequency among B cells was higher than baseline (P < 0.01). Within both PR3+ and total B cells, frequencies of transitional and naive subsets were higher at B cell recurrence than at baseline, while memory subsets were lower (P < 0.001 for all comparisons). At B cell recurrence, frequencies of B cells and subsets did not differ between patients who experienced relapse and patients who remained in remission. In contrast, the plasmablast frequency within the PR3+ B cell pool was higher in patients who experienced relapse and associated with a shorter time to relapse. Frequencies of PR3+ plasmablasts higher than baseline were more likely to be found in patients who experienced relapse within the following 12 months compared to those in sustained remission (P < 0.05). CONCLUSION: The composition of the autoreactive B cell pool varies significantly following RTX treatment in AAV, and early plasmablast enrichment within the autoreactive pool is associated with future relapses.

Serum Biomarkers of Disease Activity in Longitudinal Assessment of Patients with ANCA-Associated Vasculitis.

OBJECTIVE: Improved biomarkers of current disease activity and prediction of relapse are needed in antineutrophil cytoplasmic antibody-associated vasculitis (AAV). For clinical relevance, biomarkers must perform well longitudinally in patients on treatment and in patients with nonsevere flares. METHODS: Twenty-two proteins were measured in 347 serum samples from 74 patients with AAV enrolled in a clinical trial. Samples were collected at Month 6 after remission induction, then every 3 months until Month 18, or at the time of flare. Associations of protein concentrations with concurrent disease activity and with future flare were analyzed using mixed-effects models, Cox proportional hazards models, and conditional logistic regression. RESULTS: Forty-two patients had flares during the 12-month follow-up period, and 32 remained in remission. Twenty-two patients had severe flares. Six experimental markers (CXCL13, IL-6, IL-8, IL-15, IL-18BP, and matrix metalloproteinase-3 [MMP-3]) and ESR were associated with disease activity using all three methods (P < 0.05, with P < 0.01 in at least one method). A rise in IL-8, IL-15, or IL-18BP was associated temporally with flare. Combining C-reactive protein (CRP), IL-18BP, neutrophil gelatinase-associated lipocalin (NGAL), and sIL-2Rα improved association with active AAV. CXCL13 and MMP-3 were increased during treatment with prednisone, independent of disease activity. Marker concentrations during remission were not predictive of future flare. CONCLUSION: Serum biomarkers of inflammation and tissue damage and repair have been previously shown to be strongly associated with severe active AAV were less strongly associated with active AAV in a longitudinal study that included mild flares and varying treatment. Markers rising contemporaneously with flare or with an improved association in combination merit further study.

The association of serum interleukin-6 levels with clinical outcomes in antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: To investigate serum IL-6 (sIL-6) levels during active disease, complete remission (CR), and relapse in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), and to explore the association of changes in sIL-6 with clinical outcomes. METHODS: sIL-6 levels were measured at baseline and longitudinally over 18 months, in 78 patients with AAV enrolled in a randomized controlled trial comparing treatment with either rituximab (RTX) or cyclophosphamide (CYC)/azathioprine (AZA). Outcome variables included baseline clinical features, ANCA specificity, disease activity (active disease versus CR), time to relapse events, B cell repopulation, and ANCA titer increases. RESULTS: At baseline, sIL6 levels were detectable in 81% of patients; 73% (n = 57) of subjects were proteinase 3 (PR3)-ANCA positive, sIL-6 levels were higher in subjects with PR3-ANCAs and positively correlated with their levels (rs = 0.36,p < 0.01), but not with levels of myeloperoxidase (MPO)-ANCA (rs = -0.17,p = 0.47). Higher baseline sIL-6 levels were associated with PR3-ANCA positivity, fever, pulmonary nodules/cavities, conductive deafness, and absence of urinary red blood cell casts (p < 0.05). Baseline sIL6 levels did not predict CR at month 6 (p = 0.71), and the median sIL-6 level declined from baseline with induction therapy, regardless of CR achievement. An increase in sIL-6 during CR was a predictor for subsequent severe relapse in RTX-treated patients (hazard ratio (HR):7.24,p = 0.01), but not in CYC/AZA-treated patients (HR:0.62,p = 0.50). In contrast, a sIL-6 increase did not predict B cell repopulation or ANCA titer increase in either treatment arm (p > 0.05). CONCLUSION: At baseline, sIL-6 concentrations correlate with PR3-ANCA titers and are associated with specific clinical manifestations of AAV. Baseline sIL6 concentrations do not predict CR at 6 months, but the increase in sIL-6 concentrations during CR is associated with subsequent severe relapse among RTX-treated patients. Further investigation into the mechanistic role of IL6 in AAV might lead to identifying this pathway as a potential therapeutic target in this disease.

Association of Pulmonary Hemorrhage, Positive Proteinase 3, and Urinary Red Blood Cell Casts With Venous Thromboembolism in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: To assess the frequency of venous thromboembolism (VTE) events in the Rituximab in Antineutrophil Cytoplasmic Antibody (ANCA)-Associated Vasculitis (RAVE) trial and identify novel potential risk factors. METHODS: VTE events in 197 patients enrolled in the RAVE trial were analyzed. Baseline demographic and clinical characteristics were recorded, and univariate and multivariate analyses were performed to identify factors associated with VTE in ANCA-associated vasculitis (AAV). RESULTS: VTE occurred in 16 patients (8.1%) with an overall average time to event of 1.5 months (range 1.0-2.75). In univariate analyses with calculation of hazard ratios (HRs) and 95% confidence intervals (95% CIs), heart involvement (HR 17.408 [95% CI 2.247-134.842]; P = 0.006), positive proteinase 3 (PR3)-ANCA (HR 7.731 [95% CI 1.021-58.545]; P = 0.048), pulmonary hemorrhage (HR 3.889 [95% CI 1.448-10.448]; P = 0.008), and the presence of red blood cell casts (HR 15.617 [95% CI 3.491-69.854]; P < 0.001) were associated with the onset of VTE. In multivariate models adjusted for age and sex, the significant associations between VTE events and heart involvement (HR 21.836 [95% CI 2.566-185.805]; P = 0.005), PR3-ANCA (HR 9.12 [95% CI 1.158-71.839]; P = 0.036), pulmonary hemorrhage (HR 3.91 [95% CI 1.453-10.522]; P = 0.007), and urinary red blood cell casts (HR 16.455 [95% CI 3.607-75.075]; P < 0.001) remained. CONCLUSION: Patients diagnosed as having AAV with pulmonary hemorrhage, positive PR3-ANCA, heart involvement, and the presence of red blood cell casts are at an increased risk to develop VTE. Further studies are needed to confirm and expand these findings and to explore the mechanisms of hypercoagulability in these patients with the aim of informing potential targets for therapeutic intervention.

Disease Activity, Antineutrophil Cytoplasmic Antibody Type, and Lipid Levels in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: Patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) have an elevated risk of cardiovascular disease (CVD). This study was undertaken to develop a clearer understanding of the association between changes in disease activity and lipid levels in AAV, which may inform CVD risk stratification in this population. METHODS: Lipid levels were assessed in stored serum samples (obtained at baseline and month 6) from the Rituximab for ANCA-Associated Vasculitis (RAVE) trial, which randomized patients to receive either rituximab or cyclophosphamide followed by azathioprine. Paired t-tests and multivariable linear regression were used to assess changes in lipid levels. RESULTS: Of the 142 patients with serum samples available, the mean ± SD age was 52.3 ± 14.7 years, 72 (51%) were male, 95 (67%) were proteinase 3 (PR3)-ANCA positive, 72 (51%) had received a new diagnosis of AAV, and 75 (53%) were treated with rituximab. Several lipid levels increased between baseline and month 6, including total cholesterol (+12.4 mg/dl [95% confidence interval (95% CI) +7.1, +21.0]), low-density lipoprotein (+10.3 mg/dl [95% CI +6.1, +17.1]), and apolipoprotein B (+3.5 mg/dl [95% CI +1.0, +8.3]). These changes were observed among newly diagnosed and PR3-ANCA-positive patients but not among those with relapsing disease or myeloperoxidase-ANCA-positive patients. There was no difference in change in lipid levels between rituximab-treated patients and cyclophosphamide-treated patients. Changes in lipid levels correlated with changes in erythrocyte sedimentation rate but not with other inflammatory markers or glucocorticoid exposure. CONCLUSION: Lipid levels increased during remission induction among patients with newly diagnosed AAV and those who were PR3-ANCA positive. Disease activity and ANCA type should be considered when assessing lipid profiles to stratify CVD risk in patients with AAV.

The net effect of ANCA on neutrophil extracellular trap formation.

The formation of neutrophil extracellular traps induced by antineutrophil cytoplasmic autoantibodies has been implicated in the pathogenesis of antineutrophil cytoplasmic autoantibody-associated vasculitis. Kraaij et al. now provide evidence that excessive neutrophil extracellular trap formation in vitro induced by sera from patients with antineutrophil cytoplasmic autoantibody-associated vasculitis is associated with active disease but is not dependent on the presence of antineutrophil cytoplasmic autoantibodies.

Brief Report: Circulating Cytokine Profiles and Antineutrophil Cytoplasmic Antibody Specificity in Patients With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: To evaluate circulating cytokine profiles in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV), classified by antineutrophil cytoplasmic antibody (ANCA) specificity (proteinase 3 ANCA [PR3-ANCA] versus myeloperoxidase ANCA [MPO-ANCA]) or by clinical diagnosis (granulomatosis with polyangiitis [GPA] versus microscopic polyangiitis [MPA]). METHODS: A panel of 29 cytokines was tested in 186 patients with active AAV at inclusion into the Rituximab in AAV trial. Cytokine concentrations were compared between groups within each classification system. Multivariable analyses adjusted for age, sex, and renal insufficiency were performed, with each biomarker as a dependent variable and ANCA specificity and clinical diagnosis as explanatory variables of interest. RESULTS: Levels of 9 circulating cytokines (interleukin-6 [IL-6], granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-15, IL-18, CXCL8/IL-8, CCL-17/thymus and activation-regulated chemokine [TARC], IL-18 binding protein [IL-18 BP], soluble IL-2 receptor α [sIL-2Rα], and nerve growth factor ß [NGFß]) were significantly higher in PR3-AAV than MPO-AAV, 4 cytokines (sIL6R, soluble tumor necrosis factor receptor type II [sTNFRII], neutrophil gelatinase-associated lipocalin [NGAL], and soluble intercellular adhesion molecule 1 [sICAM-1]) were higher in MPO-AAV than in PR3-AAV, 6 cytokines (IL-6, GM-CSF, IL-15, IL-18, sIL-2Rα, and NGFß) were higher in GPA than in MPA, and 3 cytokines (osteopontin, sTNFRII, and NGAL) were higher in MPA than in GPA (all P < 0.05). For nearly all cytokines, the difference between PR3-AAV and MPO-AAV was larger than that between GPA and MPA. The multivariate analysis showed that 8 cytokines (IL-15, IL-8, IL-18 BP, NGF-ß, sICAM-1, TARC, osteopontin, and kidney injury molecule 1 (P < 0.05) distinguished patients with AAV better (lower P values and larger effect sizes) when grouped by ANCA specificity than by clinical diagnosis. CONCLUSION: Distinct cytokine profiles were identified for PR3-AAV versus MPO-AAV and for GPA versus MPA. Differences in these circulating immune mediators are more strongly associated with ANCA specificity than with clinical diagnosis, suggesting that heterogeneity in the AAV subtypes extends beyond clinical phenotypes.

Review: What Is the Current Evidence for Disease Subsets in Giant Cell Arteritis?

Giant cell arteritis (GCA) is an autoimmune vasculitis affecting large and medium-sized arteries. Ample evidence indicates that GCA is a heterogeneous disease in terms of symptoms, immune pathology, and response to treatment. In the current review, we discuss the evidence for disease subsets in GCA. We describe clinical and immunologic characteristics that may impact the risk of cranial ischemic symptoms, relapse rates, and long-term glucocorticoid requirements in patients with GCA. In addition, we discuss both proven and putative immunologic targets for therapy in patients with GCA who have an unfavorable prognosis. Finally, we provide recommendations for further research on disease subsets in GCA.

Pharmacokinetics of rituximab and clinical outcomes in patients with anti-neutrophil cytoplasmic antibody associated vasculitis.

Objectives: To study the determinants of the pharmacokinetics (PK) of rituximab (RTX) in patients with ANCA-associated vasculitis (AAV) and its association with clinical outcomes. Methods: This study included data from 89 patients from the RTX in AAV trial who received the full dose of RTX (four weekly infusions of 375 mg/m2). RTX was quantified at weeks 2, 4, 8, 16 and 24, and summarized by computing the trapezoidal area under the curve. We explored potential determinants of the PK-RTX, and analysed its association with clinical outcomes: achievement of remission at 6 months, duration of B-cell depletion and time to relapse in patients who achieved complete remission. Results: RTX serum levels were significantly lower in males and in newly diagnosed patients, and negatively correlated with body surface area, baseline B-cell count and degree of disease activity. In multivariate analyses, the main determinants of PK-RTX were sex and new diagnosis. Patients reaching complete remission at month 6 had similar RTX levels compared with patients who did not reach complete remission. Patients with higher RTX levels generally experienced longer B-cell depletion than patients with lower levels, but RTX levels at the different time points and area under the curve were not associated with time to relapse. Conclusion: Despite the body-surface-area-based dosing protocol, PK-RTX is highly variable among patients with AAV, its main determinants being sex and newly diagnosed disease. We did not observe any relevant association between PK-RTX and clinical outcomes. The monitoring of serum RTX levels does not seem clinically useful in AAV.

The Utility of Urinalysis in Determining the Risk of Renal Relapse in ANCA-Associated Vasculitis.

BACKGROUND AND OBJECTIVES: The significance of persistent hematuria or proteinuria in patients with ANCA-associated vasculitis who are otherwise in clinical remission is unclear. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A post hoc analysis was conducted using participants enrolled in two randomized, placebo-controlled clinical trials who had active GN due to ANCA-associated vasculitis, had positive ANCA, and achieved remission by month 6. Dipstick and microscopic urinalyses were performed at each visit. Persistent hematuria or proteinuria for at least 6 months and the cumulative duration of hematuria were examined. Renal relapse was defined as new or worsening red blood cell casts and/or worsening kidney function according to the Birmingham Vasculitis Activity Score for Granulomatosis with Polyangiitis. RESULTS: There were 149 patients included in this study: 42% had persistent hematuria, and 43% had persistent proteinuria beyond 6 months. Persistent hematuria was associated with a significantly higher risk of relapse, even after adjusting for potential confounders (subdistribution hazard ratio, 3.99; 95% confidence interval, 1.20 to 13.25; P=0.02); persistent proteinuria was not associated with renal relapse (subdistribution hazard ratio, 1.44; 95% confidence interval, 0.47 to 4.42; P=0.53). Furthermore, greater cumulative duration of hematuria was significantly associated with a higher risk of renal relapse (adjusted subdistribution hazard ratio, 1.08 per each month; 95% confidence interval, 1.03 to 1.12; P<0.01). The median time to renal relapse was 22 months. CONCLUSIONS: In patients with ANCA-associated vasculitis and kidney involvement who achieve remission after induction therapy, the presence of persistent hematuria, but not proteinuria, is a significant predictor of future renal relapse.

Towards precision medicine in ANCA-associated vasculitis.

ANCA-associated vasculitis (AAV) is characterized by inflammation and destruction of small and medium-sized vessels. Current management strategies for AAV have been validated in large groups of patients. However, recent insights indicate that distinct patient subsets may actually exist within AAV, thereby justifying the development of more personalized treatment strategies. In this review, we discuss current evidence for a better classification of AAV based on ANCA type. We describe how thus defined categories of AAV patients may differ in genetic background, clinical presentation, immune pathology, response to treatment and disease outcome. We also explore how these insights may provide a rationale for targeted treatments in different categories of AAV patients. Finally, we provide recommendations on how to further establish precision medicine in AAV.

Interstitial Immunostaining and Renal Outcomes in Antineutrophil Cytoplasmic Antibody-Associated Glomerulonephritis.

BACKGROUND: Immunopathologic features predict renal function at baseline and follow-up in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (GN). The interstitial infiltrate consists predominantly of T lymphocytes, but their pathophysiologic significance is unclear, especially in light of the success of B-cell-directed therapy. METHODS: Renal biopsies from 33 patients treated with cyclophosphamide (CYC; n = 17) or rituximab (RTX; n = 16) in the RTX in ANCA-associated vasculitis (RAVE) trial were classified according to the new ANCA GN classification. T- and B-cell infiltration in the interstitium was assessed by immunostaining for CD3 and CD20. Correlations of clinical and histologic parameters with renal function at set time points were examined. RESULTS: The mean (SD) baseline estimated glomerular filtration rate was 36 (20) mL/min/1.73 m2. ANCA GN class distribution was 46% focal, 33% mixed, 12% sclerotic and 9% crescentic. The interstitial infiltrate consisted of >50% CD3 positive cells in 69% of biopsies, but >50% CD20 positive cells only in 8% of biopsies. In a multiple linear regression model, only baseline glomerular filtration rate (GFR) correlated with GFR at 6, 12, and 18 months. Interstitial B- and T-cell infiltrates had no significant impact on long-term prognosis, independent of the treatment limb. A differential effect was noted only at 6 months, where a dense CD3 positive infiltrate predicted lower GFR in the RTX group and a CD20 positive infiltrate predicted higher GFR in the CYC group. CONCLUSIONS: In ANCA-associated GN, the interstitial infiltrate contains mainly T lymphocytes. However, it is neither reflecting baseline renal function nor predictive of response to treatment, regardless of the immunosuppression regimen employed.

Genetic loci of Staphylococcus aureus associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides.

The proteinase 3 (PR3)-positive anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) granulomatosis with polyangiitis (GPA) has been associated with chronic nasal S. aureus carriage, which is a risk factor for disease relapse. The present study was aimed at comparing the genetic make-up of S. aureus isolates from PR3-ANCA-positive GPA patients with that of isolates from patients suffering from myeloperoxidase (MPO)-ANCA-positive AAV, and isolates from healthy controls. Based on a DNA microarray-based approach, we show that not only PR3-ANCA-positive GPA patients, but also MPO-ANCA-positive AAV patients mainly carried S. aureus types that are prevalent in the general population. Nonetheless, our data suggests that MPO-ANCA-associated S. aureus isolates may be distinct from healthy control- and PR3-ANCA-associated isolates. Furthermore, several genetic loci of S. aureus are associated with either PR3-ANCA- or MPO-ANCA-positive AAV, indicating a possible role for pore-forming toxins, such as leukocidins, in PR3-ANCA-positive GPA. Contrary to previous studies, no association between AAV and superantigens was detected. Our findings also show that a lowered humoral immune response to S. aureus is common for PR3-ANCA- and MPO-ANCA-positive AAV. Altogether, our observations imply that the presence or absence of particular virulence genes of S. aureus isolates from AAV patients contributes to disease progression and/or relapse.

Position paper: Revised 2017 international consensus on testing of ANCAs in granulomatosis with polyangiitis and microscopic polyangiitis.

Anti-neutrophil cytoplasmic antibodies (ANCAs) are valuable laboratory markers used for the diagnosis of well-defined types of small-vessel vasculitis, including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). According to the 1999 international consensus on ANCA testing, indirect immunofluorescence (IIF) should be used to screen for ANCAs, and samples containing ANCAs should then be tested by immunoassays for proteinase 3 (PR3)-ANCAs and myeloperoxidase (MPO)-ANCAs. The distinction between PR3-ANCAs and MPO-ANCAs has important clinical and pathogenic implications. As dependable immunoassays for PR3-ANCAs and MPO-ANCAs have become broadly available, there is increasing international agreement that high-quality immunoassays are the preferred screening method for the diagnosis of ANCA-associated vasculitis. The present Consensus Statement proposes that high-quality immunoassays can be used as the primary screening method for patients suspected of having the ANCA-associated vaculitides GPA and MPA without the categorical need for IIF, and presents and discusses evidence to support this recommendation.

Are cytokines and chemokines suitable biomarkers for Takayasu arteritis?

There is a growing need for disease related biomarkers in Takayasu arteritis (TA).The assessment of pro-inflammatory cytokines and chemokines in TA may provide a better understanding of its pathophysiology, and circulating levels of these mediators may act as biomarkers of disease activity. Serum level of interleukin 6 (IL-6) is a potential biomarker for TA, which is mostly associated with TA status and disease activity. Associations between TA and serum/plasma levels of other cytokines are less clear. mRNA expression of IL-4 and tumor necrosis factor α (TNFα) are constitutively increased in peripheral blood mononuclear cells (PBMC) from TA patients and the expression of both cytokines increases even more after PBMC stimulation in vitro, while the expression of IL-10 mRNA decreases. In addition, circulating T cells from TA patients produce increased levels of both Th1- and Th17-related cytokines upon in vitro stimulation. In the aorta from TA patients, an increased expression of interferon γ (IFNγ), IL-6, IL-12 and IL-17 has been described. Regarding circulating chemokines in TA, serum/plasma levels of IL-8 (CXCL8), CCL2 and CCL5 were shown to be elevated in TA patients compared with healthy controls as well as in TA patients with active disease compared with those in remission. Serum IL-6 seems to be the best biomarker for disease state and disease activity in TA and increased Th1 and Th17 responses are predominant in the pathophysiology of TA.

Transancestral mapping and genetic load in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with marked gender and ethnic disparities. We report a large transancestral association study of SLE using Immunochip genotype data from 27,574 individuals of European (EA), African (AA) and Hispanic Amerindian (HA) ancestry. We identify 58 distinct non-HLA regions in EA, 9 in AA and 16 in HA (∼50% of these regions have multiple independent associations); these include 24 novel SLE regions (P<5 × 10-8), refined association signals in established regions, extended associations to additional ancestries, and a disentangled complex HLA multigenic effect. The risk allele count (genetic load) exhibits an accelerating pattern of SLE risk, leading us to posit a cumulative hit hypothesis for autoimmune disease. Comparing results across the three ancestries identifies both ancestry-dependent and ancestry-independent contributions to SLE risk. Our results are consistent with the unique and complex histories of the populations sampled, and collectively help clarify the genetic architecture and ethnic disparities in SLE.

M2 macrophage is the predominant phenotype in airways inflammatory lesions in patients with granulomatosis with polyangiitis.

BACKGROUND: Macrophages may present two distinct phenotypes indicated as M1 and M2 under different stimuli. M1 and M2 macrophages have divergent functions that range from enhancement of inflammation for M1 to tissue repair and remodeling for M2 macrophages. The objective of this study was to evaluate the distribution of M1 and M2 macrophage phenotypes in biopsies from the airways of patients with active granulomatosis with polyangiitis (GPA) and to analyze their associations with T and B cells in those biopsies, and with nasal carriage of Staphylococcus aureus, disease parameters and therapy. METHODS: Consecutive GPA patients (n = 35) with active airway disease, who underwent respiratory tract biopsy were included. Immunohistochemical evaluation was performed to assess the distribution of macrophages and T and B cells using the markers CD68, CD3 and CD20, respectively. CD86 was used as the M1 marker and CD163 as the M2 marker while Tbet and GATA-3 were used as Th1 and Th2 markers, respectively. At the time of the biopsy patients were assessed for nasal carriage of Staphylococcus aureus and treatment. RESULTS: Percentages of macrophages and T cells were significantly higher than those of B cells in lesional tissue from the respiratory tract in GPA. M2 macrophages and Th2 cells were more frequent than M1 macrophages (p = 0.0007) and Th1 cells (p < 0.0001), respectively. Percentages of T cells were higher in nose biopsies than in biopsies from other sites (p = 0.021); macrophages and CD163+ macrophages were more predominant in biopsy sites other than the nose (p = 0.039 and p = 0.012, respectively). Carriage of Staphylococcus aureus was associated with higher T cell scores (p = 0.014). The frequency of macrophages, especially M2 macrophages, was higher in GPA patients treated with immunosuppressive agents (p = 0.010); daily prednisolone dose was positively correlated with all macrophage markers. However, in multivariate analysis no independent associations were found between disease parameters and therapy with macrophage markers or T cells. CONCLUSION: In GPA, M2 is the predominant macrophage phenotype in the respiratory tract. Although some associations were observed between macrophages and T cells with therapy and nasal carriage of Staphylococcus aureus, they were not independently significant in multivariate analysis.

Association of Serum Calprotectin (S100A8/A9) Level With Disease Relapse in Proteinase 3-Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: S100A8/A9 (calprotectin) has shown promise as a biomarker for predicting relapse in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This study was undertaken to investigate serum S100A8/A9 level as a biomarker for predicting future relapse in a large cohort of patients with severe AAV. METHODS: Serum levels of S100A8/A9 were measured at baseline and months 1, 2, and 6 following treatment initiation in 144 patients in the Rituximab in ANCA-Associated Vasculitis trial (cyclophosphamide/azathioprine versus rituximab [RTX] for induction of remission) in whom complete remission was attained. RESULTS: Patients were divided into 4 groups: proteinase 3 (PR3)-ANCA with relapse (n = 37), PR3-ANCA without relapse (n = 56), myeloperoxidase (MPO)-ANCA with relapse (n = 6), and MPO-ANCA without relapse (n = 45). Serum S100A8/A9 level decreased in all groups during the first 6 months of treatment. The percentage reduction from baseline to month 2 was significantly different between patients who experienced a relapse and those who did not in the PR3-ANCA group (P = 0.046). A significantly higher risk of relapse was associated with an increase in S100A8/A9 level between baseline and month 2 (P = 0.0043) and baseline and month 6 (P = 0.0029). Subgroup analysis demonstrated that patients treated with RTX who had increased levels of S100A8/A9 were at greatest risk of future relapse (P = 0.028). CONCLUSION: An increase in serum S100A8/A9 level by month 2 or 6 compared to baseline identifies a subgroup of PR3-ANCA patients treated with RTX who are at higher risk of relapse by 18 months. Since RTX is increasingly used for remission induction in PR3-ANCA-positive patients experiencing a relapse, S100A8/A9 level may assist in identifying those patients requiring more intensive or prolonged treatment.